Nearly all disease states in multicellular organisms involve the action of proteins. Classic therapeutic approaches have focused on the interaction of proteins with other molecules in efforts to moderate the proteins disease-causing or disease-potentiating activities. In newer therapeutic approaches, modulation of the production of proteins has been sought. A general object of some current therapeutic approaches is to interfere with, or otherwise modulate, gene expression.
One method for inhibiting the expression of specific genes involves the use of oligonucleotides, particularly oligonucleotides that are complementary to a specific target messenger RNA (mRNA) sequence, known as antisense oligonucleotides. Several oligonucleotides are currently undergoing clinical trials for such use. Phosphorothioate antisense oligonucleotides are presently being used as antiviral agents in human clinical trials.
Oligonucleotides and their analogs can be designed to have particular properties. A number of chemical modifications have been introduced into oligomeric compounds to increase their usefulness as therapeutic agents. Such modifications include those designed to increase binding affinity to a target strand, to increase cell penetration, to stabilize against nucleases and other enzymes that degrade or interfere with the structure or activity of the oligonucleotide, to provide a mode of disruption (terminating event) once the oligonucleotide is bound to a target, and to improve the pharmacokinetic properties of the oligonucleotide. Despite such modifications, the cellular uptake of oligomeric compounds remains poor.
Oligonucleotides have been formulated with various with transfection agents, including anionic and cationic lipids and polyamines, in an attempt to improve their ability to permeate biological membranes. Dheur, S.; Saison-Behmoaras, T. E. Methods Enzymol. 2000, 313, 56-73; Vinogradov, S.; Batrakova, E.; Kabanov, A. V. Polym. Prepr. (Am. Chem. Soc., Div. Polym. Chem.) 2000, 41, 1641-1642; Vinogradov, S.; Batrakova, E.; Kabanov, A. Colloids Surf., B 1999, 16, 291-304; Bandyopadhyay, P.; Ma, X.; Linehan-Stieers, C.; Kren, B. T.; Steer, C. J. J. Biol. Chem. 1999, 274, 10163-10172; Auvray, P.; Sourdaine, P.; Seralini, G. E. Biochem. Biophys. Res. Commun. 1998, 253, 1-9; Demeneix, B. A.; Boussif, O.; Zanta, M. A.; Remy, J. S.; Behr, J. P. Nucleosides Nucleotides 1997, 16, 1121-1127. Of the transfection agents used, polyethylenimines (PEI) are generally the most efficient and least expensive delivery vehicles. Kren, B. T.; Parashar, B.; Bandyopadhyay, P.; Chowdhury, N. R.; Chowdhury, J. R.; Steer, C. J. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 10349-10354. It was observed, however, that, although complexes of excess PEI and oligonucleotide phosphorothioates were efficiently taken up by cells, the oligonucleotides failed to dissociate in the cytoplasm, resulting in no appreciable enhancement in the antisense activity of the oligonucleotides. Dheur, S.; Dias, N.; Van Aerschot, A.; Herdewijn, P.; Bettinger, T.; Remy, J.-S.; Helene, C.; Saison-Behmoaras, E. T. Antisense Nucleic Acid Drug Dev. 1999, 9, 515-525.
A need therefore exists in the art for the development of means to improve the cellular uptake and cellular distribution of oligomeric compounds.